Bioremediation potential of consortium Pseudomonas Stutzeri LBR and Cupriavidus Metallidurans LBJ in soil polluted by lead

Pollution by lead (Pb) is an environmental and health threat due to the severity of its toxicity. Microbial bioremediation is an eco-friendly technique used to remediate contaminated soils. This present study was used to evaluate the effect of two bacterial strains isolated and identified from Bizerte lagoon: Cupriavidus metallidurans LBJ (C. metallidurans LBJ) and Pseudomonas stutzeri LBR (P. stutzeri LBR) on the rate of depollution of soil contaminated with Pb from Tunisia. To determine this effect, sterile and non-sterile soil was bioaugmented by P. stutzeri LBR and C. metallidurans LBJ strains individually and in a mixture for 25 days at 30°C. Results showed that the bioaugmentation of the non-sterile soil by the mixture of P. stutzeri LBR and C. metallidurans LBJ strains gave the best rate of reduction of Pb of 71.02%, compared to a rate of 58.07% and 46.47% respectively for bioaugmentation by the bacterial strains individually. In the case of the sterile soil, results showed that the reduction rate of lead was in the order of 66.96% in the case of the mixture of the two bacterial strains compared with 55.66% and 41.86% respectively for the addition of the two strains individually. These results are confirmed by analysis of the leachate from the sterile and non-sterile soil which showed an increase in the mobility and bioavailability of Pb in soil. These promising results offer another perspective for a soil bioremediation bioprocess applying bacterial bioremediation.


Introduction
Over recent decades, the mining industry has played an overwhelming role in global energy and has generated significant economic benefits annually for several regions of the country [1]. However, mining is one of the industrial activities that causes the largest and most persistent alterations to the ecosystem and human health [2], including loss of biodiversity and accumulation of pollutants in the environment. It represents one of the main sources of heavy metals and metalloids [3] pollution in the soil, due to the large quantities of discharges with high levels of toxic chemicals [4] and stocks that are often abandoned without treatment [3]. a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 bioremediation, recovery and limitation of heavy metals [18]. It carried many determinants of heavy metal resistance implicated in different mechanisms, including efflux systems, complexation, reduction, and reductive precipitation, which enable cell detoxification and survival [22]. In our study, we used the bioaugmentation process, which consists of the addition of precultured microbial cultures (individually or in consortium) P. stutzeri LBR and C. metallidurans LBJ, with a high power to degrade pesticides, hydrocarbons and heavy metals, in order to improve the degradation of pollutants. This process is one of the most common and bestsuited approaches for bioremediation of polluted sites and remediation of in situ contaminants with minimal impact on surface ecosystems [23]. Compared to ex-situ bioremediation techniques such as pumping, biopile and primary treatment, bioaugmentation is a low-cost, environmentally friendly method that generates little waste after treatment. The main objectives of this research were: i. To study the effectiveness of bacteria P. stutzeri LBR and C. metallidurans LBJ in remediating Pb-contaminated soil through the use of the microbial bioremediation process of bioaugmented bacterial strains individually and in consortium, ii. To follow the rate of reduction of Pb concentration as a function of bacterial growth kinetics while analyzing the concentration of Pb in the leachate (the medium in which the soil bathes throughout the bioremediation experiment).

Study area and soil samples
Soil samples were taken from a highly polluted area with heavy metals; it is a mining area located in Tunisia. Samples were collected from 0 to 20 cm below the soil surface [10], by quincunx sampling method, then they were sieved (mesh size two mm), mixed, and transported to the laboratory in sterile bottles at 4˚C, in accordance with the ISO 19458 sampling standard.

Dosage of heavy metals
The dosage of heavy metals in the soil sample was determined using Inductively Coupled Plasma Optical Emission Spectrophotometry ICP-OES (PerkinElmer Optima 7300 DV, Inc., Shelton, CT, USA) in accordance with the requirements of ISO 11885, March 1998.

Bacterial strains used in bioremediation by bioaugmentation of soil contaminated with heavy metals
The bacterial strains used in bioremediation by bioaugmentation of soil contaminated with Pb are: P. stutzeri LBR strain and C. metallidurans LBJ strain, isolated from sediment of Bizerte lagoon in our Laboratory of Biochemical and Molecular Biology in the Faculty of Sciences of Bizerte, Tunisia in the work of Mansouri and colleagues [18]. The sequences of these bacterial strains P. stutzeri LBR and C. metallidurans LBJ have been deposited in the GenBank database [18]. These isolated bacterial strains were transplanted to the PCA culture medium and incubated at 37˚C for 24-48 hours. After incubation, these strains were maintained on glycerol at -80˚C for long-term conservation. The morphological characterization and Gram staining of the bacterial strains P. stutzeri LBR and C. metallidurans LBJ proves that both strains are bacilli Gram (-), of Star shape with irregular contour (umbilical) and pink colour for the strain P. stutzeri LBR, and of regular Circular shape of White contour with Yellow point for the strain C. metallidurans LBJ. In terms of biochemical characterization, the strain P. stutzeri LBR gave positive reaction for oxidase activity, coagulase activity, fermentation of glucose, lactose and negative reaction for catalase activity, H 2 S, Gas, and mannitol. The C. metallidurans LBJ strain characterized by a positive reaction for catalase activity, fermentation of glucose, lactose, H 2 S + , Gas + and a negative reaction for oxidase activity, coagulase activity and mannitol [18].

Bioaugmentation tests of contaminated soil sample
For bioaugmentation tests in the sterile and non-sterile soil, we followed the same steps except, in the case of the sterile soil, sterilizing it by autoclaving at 120˚C for 15 min [16]. In a flask, 10 g of soil was mixed with 500 mL of sterilized M9 medium, then 70 All treatments were performed in triplicate. Every five days the bacterial counts and analysis of Pb by ICP-OES was realized. All necessary aseptic conditions were respected.

Statistical analysis
Means and standard deviations were calculated using STATISTICA 8.0. All analyses were performed three times. The comparative analysis of the data was determined with an analysis of variance test (ANOVA) with significant differences at p<0.05. R Foundation for Statistical Computing Version 3.6.3 statistical software was used for testing (R Development Core Team, 2009-2018 RStudio, Inc.).

Study of the physicochemical parameters of the soil sample
The results for physicochemical characteristics showed that the pH measured in soil showed an acidic pH of about 5.09 at a temperature of 17.70˚C. This result promotes the trophic transfer of heavy metals [25]. The electrical conductivity of the polluted soil shows a value of 603.06 μS/cm. This value can describe a very low salinity of the order of 0.22. Another value, the percentage of dry matter and soil humidity, shows that it is a soil rich in dry matter (96% DM) and low humidity 2%.

Dosage of heavy metals in soil sample
The analysis of the metal elements in the polluted soil sample by ICP shows a significant increase in the concentrations of heavy metals in the soil compared to the standard set by the European Directive. In particular, Pb has the highest concentration, which is in the order of 896.84 ± 0.04, three times higher than the standard of the European Directive (50 to 300). This means that the soil is highly saturated with lead. Cadmium also exceeds the European standard set with a concentration of 18.84 ± 0.03. The other values do not present a serious environmental problem as shown in Table 1.

Soil remediation study by microbial bioremediation in sterile soil
The result of bioaugmentation of sterile soil by the bacterial strains P. stutzeri LBR and C. metallidurans LBJ are presented in Fig 1. To monitor the rate of Pb reduction, we have tracked the growth kinetics of these bacterial strains over 25 days at 30˚C.
In the first 15 days of the experiment (phase 1), we observed that the increase in the bacterial growth rate of the strains P. stutzeri LBR and C. metallidurans LBJ in consortium led to a significant reduction (p< 0.05) of Pb concentration in sterile soil.
Under these conditions, the Pb concentration fell from 800.05 mg/kg to 264.41 mg/kg within 25 days at 30˚C, which corresponds to a percentage reduction of 66.96%. During this phase, the number of bacteria in the consortium reached a peak of 65.109 CFU/mL, followed by a decrease in the number of cells, which reached 25.109 CFU/mL at the end of the experiment.
In the sterile soil bioaugmented with the P. stutzeri LBR strain, we found that the total bioaugmented soil flora showed growth during the first 15 days (phase 1), followed by a decrease in the number of cells at the end of the experiment. This is accompanied by a significant reduction in the Pb concentration from 801.08 mg/kg to 355.18 mg/kg over 25 days, with a reduction percentage of 55.66%. The increase in bacterial growth rate of the individually bioincreased C. metallidurans LBJ strain during the first phase of the experiment resulted in a significant reduction in Pb concentration from 800.41 mg/kg to 465.37 mg/kg over 25 days, with a reduction percentage of 41.86%. Beyond this phase, the number of cells of the C. metallidurans LBJ strain decreased. Therefore, the sterile soil bioaugmented with the consortium showed a significant decrease in Pb concentration compared to the sterile soil bioaugmented with individual strains. These results are confirmed by the statistical analysis on the R software showing that there is a significant difference between the three Pb reduction rates obtained under the different conditions Pb SS1, Pb SS2 and Pb SS3.

Leachate analysis after sterile soil bioaugmentation
To verify the effect of the bacterial strains used on the reduction of the concentration of Pb in soil, an analysis of the leachate of the sterile soil (the medium in which the soil bathes throughout the experiment) was carried out throughout the bio augmentation experiment. Therefore, there is a significant difference (p<0.05) between sterile soil bioaugmented with the bacterial consortium and individually bioaugmented soil compared to the control.

Soil remediation study by microbial bioremediation in non-sterile soil
Another experiment in the bioaugmentation of non-sterile soil with the bacterial strains P. stutzeri LBR and C. metallidurans LBJ was realized to track the rate of Pb reduction as a function of bacterial growth over 25 days at 30˚C (Fig 3).
This figure shows that the growth rate of the entire bacterial population in the presence of P. stutzeri LBR and C. metallidurans LBJ strains in consortium in non-sterile soil increased during the first 20 days of the experiment (phase 1) from 20.109 CFU/mL to 70.109 CFU/mL. This increase resulted in a significant reduction (p<0.05) in Pb concentration from 801.33 mg/kg to 232.19 mg/kg over 25 days at 30˚C, with a percentage reduction of 71.02%. Beyond this phase, the total bacterial count decreased. The increase in growth rate of the entire bacterial population with the individually bioaugmented P. stutzeri LBR strain in the non-sterile soil during Phase 1 resulted in a significant reduction in Pb concentration. Under these conditions, the Pb concentration decreased from 801.28 mg/kg to 335.91 mg/kg over 25 days, corresponding to a reduction of 58.07%. In non-sterile soil bioaugmented with strain C. metallidurans LBJ, the number of the whole bacterial population of the bioaugmented soil showed an increase during the first 20 days (phase 1), followed by a decrease in the total number of bacterial cells at the end of the experiment. This is accompanied by a significant reduction in the Pb concentration from 799.74 mg/kg to 428.07 mg/kg over 25 days, with a percentage reduction of around 46.47%. Therefore, the bioaugmented non-sterile soil with the consortium showed a significant decrease in Pb concentration compared to the individually bioaugmented soil with the P. stutzeri LBR and C. metallidurans LBJ strains. These results are confirmed also by the statistical analysis showing that there is a significant difference between the three Pb reduction rates obtained under the different conditions Pb NS 1, Pb NS 2 and Pb NS 3.

Leachate analysis after non-sterile soil bioaugmentation
Similar to the sterile soil, a leachate analysis of the non-sterile soil (the environment in which the soil bathes throughout the experiment) was conducted throughout the bioaugmentation experiment to explain the effect of the entire bacterial population of non-sterile soil with the P. stutzeri LBR and C. metallidurans LBJ strains used, on the reduction of Pb concentration. The change in the concentration of Pb in the leachate of non-sterile soil bioaugmented with bacterial strains over time is shown in Fig 4. From this figure we noted that during the 25 days of the experiment, the concentration of Pb in the leachate of the non-sterile soil bioaugmented with the bacterial strains increased significantly compared to the control (C.NS). Indeed, the concentration of Pb increased from 0.29 mg/L to 19.73 mg/L in the leachate of the bioaugmented soil with the consortium, from 0.28 mg/L to 15.98 mg/L in the soil bioaugmented with the P. stutzeri LBR strain, and from 0.25 mg/L to 12.92 mg/L in the soil bioaugmented with the C. metallidurans LBJ strain throughout the experimental period. Therefore, there is a significant difference (p<0.05) between the non-sterile soil bioaugmented with the bacterial consortium and the individually bioaugmented soil compared to the control.

Discussion
This study was based on the microbial bioremediation of sterile and non-sterile polluted soil, contaminated with heavy metals, in particular Pb. For soil bioremediation, we used two bacterial strains P. Stutzeri LBR and C. metallidurans LBJ [18]. Depollution efficiency is influenced by the resistance capacity of P. Stutzeri LBR and C. metallidurans LBJ bacteria to Pb through their ability to adsorb and accumulate metal ions [26].
This work began with a physicochemical characterization of the soil sample. The pH analysis of the polluted soil showed an acidic pH. This result is in agreement with the work of Serpaud and colleagues [27] that showed that Pb tends to predominate in an acid medium (pH = 5.6). Indeed, the biosorption capacity of Pb on the bacterial cell surface is strongly dependent on increasing pH from 2 to 6, with a maximum uptake capacity at pH 5 [28]. Moreover, Sharma and colleagues [42] showed that pH can have a significant effect in helping bacteria to mobilize certain metals at pH 6.2 to 8. A series of bioaugmentation experiments was carried out over 25 days in sterile and non-sterile soil.
These tests allowed us to study the evolution of the rate of reduction in Pb concentration according to the kinetics of bacterial growth. The results showed that the increase in the bacterial growth rate led to a reduction in the concentration of Pb in the soil especially during phase 1 of the experiment. Beyond this phase, the decrease in the number of bacterial cells in the sterile soil and non-sterile soil may be due to the depletion of the carbon source (glycerol) added to the medium. The best Pb concentration reduction rate was observed in non-sterile soil bioaugmented with the consortium of P. Stutzeri LBR and C. metallidurans LBJ strains, with a value of 71.02% compared to a rate of 66.96% in the case of sterile soil bioaugmented with the consortium. These results showed that the bacterial consortium of the strains P. stutzeri LBR and C. metallidurans LBJ are characterized by a high efficiency of lead biosorption compared to the work of Waite and colleagues [29], who proved that the strain P. stutzeri W228 presents significant results for the absorption of Pb going to concentrations up to 54.0 mg/kg. Further, the work of Ayangbenro and Babalola [30] found that the strain of Bacillus cereus NWUAB01 isolated from mining soil showed a lead removal efficiency of 69%.
The increase in the rate of Pb reduction in non-sterile soil compared to sterile soil is apparently due to the combined action between the microbial population already present in the soil and the bioaugmented bacteria. In addition, the combined action of the consortium formed by the bacterial strains P. Stutzeri LBR and C. metallidurans LBJ in both the sterile and non-sterile soil showed a synergistic effect which was manifested by a rate of Pb reduction greater than that obtained in the case of soil bioaugmented with bacterial strains individually.
This result is consistent with the work of Diels and colleagues [31], who showed that the combination of resistance to heavy metals improves the bioprecipitation capacity of metals. Therefore, bacterial mixtures are proven to be the most effective microbial bioremediation method, compared to single-strain culture methods, which could be due to higher bacterial cell density. This hypothesis is confirmed by the work of Kang and colleagues [32], who concluded that the isolation and mixing of native bacteria (Viridibacillus arenosi, Sporosarcina soli, Enterobacter cloacae and E. cloacae) in the bioremediation process lead to increased growth rates and higher resistance to heavy metals than individual cultivation methods.
P. stutzeri LBR has preferential biosorption to Pb, with inertization of more toxic metals (Pb) compared to less toxic metals (Cu) [21]. This conclusion explained the result that the greater rate of Pb reduction appeared in the sterile and non-sterile soil bioaugmented only with the P. Stutzeri LBR strain respectively of 55.66% and 58.07%.
As a result, bacterial cells invest in the production of exopolysaccharides (ESP) (involved in metal resistance mechanisms [33]) which facilitates the biosorption process of Pb by P. stutzeri LBR by the existence of ionizable functional groups, such as carboxyl (COOH), phosphate (RHPO 4 ), hydroxyl (R-OH), sulfhydryl (R-SH), phenolic (R C 6 H 5 OH) and amine (R-NH 2 ), that permit them to bind toxic heavy metals, particularly Pb [34,35]. Likewise, the enzymatic activities of the ESP contribute to the inertization and detoxification of Pb via precipitation and transformation processes at the level of the polymer mass, creating a biosorbent agent that is particularly ideal for bioremediation due to their involvement in the flocculation process and the binding of metal ions from solutions [33,36,37]. Also, the structure and composition of the ESP varied according to the phase of bacterial growth, where a greater decrease of lead concentration was observed especially during phase 1 of growth, in sterile and non-sterile soil, due to the high incorporation of acidic sugar in the ESP [38].
The low Pb reduction rate in the sterile and non-sterile soil bioaugmented only with the strain C. metallidurans LBJ (41.86% and 46.47% respectively) compared to the Pb rate reduction in the sterile and non-sterile soil bioaugmented with P. stutzeri LBR only is explained by the higher affinity of C. metallidurans LBJ to Cadmium compared to Pb and thereby an average capacity of biosorption to some other metals despite being a multi-resistant bacterial model [39]. However, this reduction is explained by the ability of Pb to accumulate in polyphosphate inclusions [40]. Indeed, C. metallidurans LBJ has the pbrABCD operon which encodes the pbrD protein involved in resistance to Pb. This is an intracellular protein which allows the sequestration of Pb using a binding site rich in residues of cysteine, proline and serine, which reduces its toxic effects [41].
To better check the efficacy of the bacterial strains used P. stutzeri LBR and C. metallidurans LBJ in the bioremediation process to reduce the concentration of Pb in polluted soil, a leachate analysis was conducted for the bioaugmented sterile and non-sterile soils. A clear increase in the concentration of Pb in the leachate was observed, which proves that the bacteria P. stutzeri LBR and C. metallidurans LBJ raised the mobility and solubility of Pb in the soil. As a consequence, the bioavailability of this metal is increased, which required its transfer from the soil to the liquid fraction so that it can be easily precipitated and removed by the leaching process [33]. These results are consistent with the work of Reith and colleagues [42], who proved that microorganisms are the main drivers of heavy metal mobility. The bacteria P. Stutzeri LBR and C. metallidurans LBJ can therefore solubilize metals by production of siderophores and then adsorb metals on their biomass, on the external membrane proteins induced by the metals and by bioprecipitation. Of which, a relationship was observed between the bioavailability of heavy metals and the overproduction of siderophores [31].

Conclusion
The results obtained in this work showed that microbial bioremediation with the strains P. Stutzeri LBR and C. metallidurans LBJ on the reduction of the concentration of Pb, in sterile and non-sterile polluted soil, can be considered as an effective method of environmental protection. A significant reduction in the concentration of Pb was particularly exhibited in the bioaugmented non-sterile (71.02%) and sterile (66.96%) soil in the presence of the consortium of both considered bacterial strains together. These two reduction rates in the concentration of Pb are still higher than that found in the soil bioaugmented with the bacterial strains P. Stutzeri LBR and C. metallidurans LBJ, separately. These results are confirmed by the analysis of sterile and non-sterile soil leachate, which proves an increase in the solubility, mobility and bioavailability of Pb from the solid fraction to the liquid fraction following an increase in the concentration of this metal in the leachate. In conclusion, the microbial bioremediation of polluted soil contaminated with Pb, by the strains P. stutzeri LBR and C. metallidurans LBJ, yielded very satisfactory results, hence the effectiveness of this eco-friendly biological process to clean up soils contaminated with heavy metals.
Supporting information S1 Table. Cell growth of bacterial strains P. stutzeri LBR and C. metallidurans LBJ in sterile soil during bioaugmentation.